In recent years, coronary artery disease (CAD) has become a major cause of death in western societies. Hyperlipidemia and atherosclerosis are regarded as major risk mechanisms for coronary diseases, whereas there is some evidence that hypertension, diabetes and excess body weight may have less negative impact on coronary disorders than previously assumed.
Atherosclerosis is thought to be initiated at critical sites of the arterial vasculature by a process of monocyte adhesion to the vessel wall, sustained by the occurrence of active functional changes on the endothelial surface (M. A. Gimbrone et al., Thromb. Haemost. 82(2) 722-6 (1999)).
Blood platelets play a major role in coronary artery disease (B. A. Osterud, Thromb. Res. 85: 1-22 (1997)). Platelets are found at the sites of early atherosclerotic lesions. When activated, platelets secrete potent mitogenic factors such as platelet-derived growth factor, transforming growth factor β, and epidermal growth factor, which lead to smooth muscle proliferation and progression of atherosclerotic lesions. Enhanced platelet reactivity and spontaneous platelet aggregation were associated with a higher risk of recurrent coronary artery disease. Physiologic anti-platelet metabolites, such as nitric oxide, activate platelet guanylate cyclase and elevate cyclic guanosine-3′,5′-monophosphate, thereby reducing fibrinogen binding to the glycoprotein IIb-IIIa (GPIIb-IIIa) receptor through inhibition of agonist-mediated calcium flux.
Several interventions demonstrated a decreased risk of cardiovascular disease, such as with therapeutic doses of aspirin (R. Collins et al., N. Eng. J. Med. 336: 847-60 (1997)) and antioxidant supplements (C. H. Hennekens, Am. Heart J. 128: 1333-6 (1994)). Therapy with anti-platelet agents such as aspirin and clopidogrel significantly decreases the incidence of primary and secondary coronary events (C. H. Hennekens, Annu. Rev. Public Health 18: 37-49 (1997)). Antibodies and peptides that block the fibrinogen binding to activated platelet glycoprotein IIb-IIIa have improved the results of coronary revascularisation procedures. Activation-dependent platelet antigens also indicate changes in platelet function after physical exercise, physiologic challenges (D. Rajasekhar, Thromb. Haemost. 77: 1002-7 (1997)), and dietary intervention (M. A. Allman-Farinelli et al., Thromb. Res. 90: 163-9 (1998)).
In recent years, certain dietary components, notably unsaturated fatty acids and polyphenols, have been identified as key mediators in numerous cellular processes. As observed by De Caterina et al. (Atherioscler. Thromb. 14: 1829-36 (1994); Prostaglandins Leukot. Essent. Fatty Acids 52: 191-5 (1995)), ω-3 fatty acids, which have effects on total and LDL cholesterol and yet also appear to be linked to protection from atherosclerosis, may act by inhibiting early atherogenic events related to monocyte adhesion to endothelial cells. This process occurs through inhibition of endothelial activation, i.e. the concerted expression of cytokine-inducible endothelial leukocyte adhesion molecules and leukocyte chemo-attractants affecting monocyte adhesion. Inhibition of a common signal-transduction pathway involving the transcription factor nuclear factor-κB (NF-κB) was therefore suggested.
The involvement of nuclear factor-κB has been established by M. A. Carluccio et al. (Atherioscler. Thromb. Vasc. Biol. 23(4): 622-629 (2003)). They demonstrated that oleuropein and hydroxytyrosol (major components of olive leaf extract) inhibit at nutritionally relevant concentrations transcriptional endothelial adhesion molecule expression, thus possessing atheroprotective features.
Several epidemiological and laboratory studies indicate that moderate alcohol consumption can lower the risk of atherosclerosis and hyperlipidemia and thus the chance of coronary artery disease (E. B. Rimm et al., BMJ 312: 731-36 (1996); D. M. Goldberg et al., Clin. Chim. Acta 237: 155-187 (1995)). In France, where the red wine consumption is relatively high as compared to Northern European Countries and the USA, but the pattern of saturated fat intake similar, the mortality rate of CAD is approximately 50% lower. This so called “Trench Paradox” (S. Renaud et al., Lancet 339: 1523-1526 (1992)) has been attributed to the major constitute of “wine tannins” the polyphenolic alcohols, such as gallic acid and flavonoids of the catechin family including (+)-catechin, (−)-epicatechin, and procyanidin B2, which are abundantly present in red wine (S. Rosenkranz et al., Faseb J. 16: 1958-1976 (2002); P. M. Kris-Etherton et al., Am. J. Med. 113, Suppl. 9B: 71-88 (2002)).
Phenolic substances contained in red wine have been found to inhibit oxidation of human LDL and thus postulated to possess atheroprotective properties. However, it is difficult to attribute a reduction of atherosclerotic processes and consequently protection from coronary artery disease only to the inhibition of LDL oxidation because many vascular effects of antioxidants are not related to the resistance of LDL to oxidation (M. N. Diaz et al., N. Eng. J. Med. 337: 408-416 (1997)).
Several studies carried out in humans and animals have shown that wine phenolic compounds could exert their effects by reducing prostanoid synthesis from arachidonate. In addition, it has been suggested that wine phenolic fractions could reduce platelet activity mediated by nitric oxide. Moreover, wine phenolic components increase vitamin E levels, while decreasing the oxidation of platelets submitted to oxidative stress.
M. E. Ferrero et al. (Am. J. Clin Nutr. 68: 1208-14 (1998)) showed the role of resveratrol (a polyphenol present in red wine) in the regulation of the endothelial cell adhesion molecule-1 expression, and thus demonstrated that its antiatherogenic activity is not in the front rank related to the protection of LDL from oxidation as postulated by M. N. Diaz et al. (ibid).
S. Rotondo et al. (Br. J. Pharmacol. 123(8): 1691-99 (1998)) investigated the effect of trans-resveratrol on functional and biochemical responses of polymorphonuclear leukocytes (PMN), which are suggested to be involved in the pathogenesis of acute coronary heart diseases. The results of their studies indicate that trans-resveratrol interferes with the release of inflammatory mediators by activated PMN and down-regulates adhesion-dependent thrombogenic PMN functions.
M. A. Carluccio et al. (Atherioscler. Thromb. Vasc. Biol. 23(4): 622-29 (2003)) confirmed recently the conclusions by Ferrero et al. and S. Rotondo et al. establishing the involvement of nuclear factor-κB as key transcription factor for the endothelial cell adhesion molecule-1 expression.
D. Rein et al. (Am. J. Clin. Nutrition 72(1): 30-35 (2000); J. of Nutrition 130 (8S): 2120S-2126S (2000)) executed a series of in vitro and in vivo studies on the effects of cocoa procyanidins (trimers and pentamers), epicatechin and de-alcoholised wine (DRW) on platelet activation. Fluorescent-labeled monoclonal antibodies recognizing the fibrinogen binding conformation of GPIIb-IIIa (PAC-1 binding) and the activation-dependent platelet epitope CD62P (P-selectin) were selected as markers for the platelet activation. Both tested components added to whole blood in vitro increased PAC-1 binding and P-selectin expression on unstimulated platelets, but suppressed the platelet activation in response to epinephrine. In contrast, cocoa procyanidins inhibited stimulated platelet activation in whole blood, whereas the effect of de-alcoholised wine was not that much pronounced. Generally, this suppressive effect observed on platelet reactivity may explain the cardioprotective effects of polyphenols present in wine or other nutritional preparations (e.g. cocoa beverages and chocolate).
Red wine polyphenols and their impact on platelet aggregation have been moreover studied by P. Russo et al. (Nutr. Metab. Cardiovasc. Dis. 11(1): 25-9 (2001)). They have isolated four classes of wine phenolic compounds: phenolic acids (fraction 1), procyanidins, catechins and monomeric anthocyanidins (fraction 2), flavonols and resveratrol (fraction 3) and polymeric anthocyanidins (fraction 4). The effect of each fraction on ADP-induced platelet aggregation in rats and c-AMP content was compared with that of de-alcoholised red wine (DRW) and the pure phenolic compounds alone (quercetin, catechin, resveratrol, caffeic acid). Both DRW and the phenolic fraction 2 inhibited significantly ADP-induced platelet aggregation, whereas the effects of fractions 3 and 4 and the pure phenolic compounds were not significant. A significant increase in platelet c-AMP content was observed first after the addition of DRW and fraction 2.
As another approach to exhibit the impact of polyphenols in red wine on the platelet activation and consequently their atheroprotective properties, A. D. Blann et al. (Blood Coagul. Fibrinolysis 13(7): 647-651 (2002)) explored markers of platelet activity (beta-thromboglobulin and soluble P-selectin) and endothelial cell function (von-Willebrand-factor and soluble thrombomodulin) before and upon ingestion of red wine in vivo. The only significant increase was noticed for beta-thromboglobulin. The study results led to the conclusion that red wine activates the platelets without having any substantial effect on the endothelium.
Rosenkranz et al. (Faseb J. 16: 1958-1976 (2002)) observed that non-alcoholic constituents of red wine that accumulate during mash fermentation act as potent inhibitors of platelet-derived growth factor β (βPDGFR) signalling and PDGF-dependent cellular responses in vascular smooth muscle cells (VSMC). Signals initiated by the βPDGFR play an important role in vascular development and the pathogenesis of atherosclerosis. PDGF-dependent migration and proliferation of VSMC are critical steps during atherogenesis. In the same work they demonstrated that mainly the flavonoids of the catechin family inhibit the PDGF-dependent tyrosine phosphorylation of the βPDGFR, whereas gallic acid only does not mediate a significant effect.
Furthermore, it is known from U.S. Pat. No. 6,133,311 that gallic acid inhibits the activities of 3-hydroxy-3-methyl-glutaryl-Coenzyme A (HMG-CoA reductase), which mediates the synthesis of mevalonic acid, an intermediate in the biosynthesis of sterols, e.g. cholesterol, or isoprenoids (William W. Parmley and Kanu Chatterjee (Eds.), Cardiovascular Pharmacology, Wolfe Publishing, 1994). Thus, it reduces the rate of cholesterol biosynthesis and prevents therefore arteriosclerosis and hypercholesterolemia which are known to be strongly related to CAD.
In U.S. Pat. No. 6,133,311, an additional mechanism of action is proposed for gallic acid when used to control increased plasma cholesterol level. They are referring to an interaction of gallic acid with the acyl CoA-cholesterol-o-acyltransferase (ACAT). ACAT promotes the esterification of cholesterol in blood. Foam cells are formed by the action of ACAT and contain a large amount of cholesterol ester carried by low density lipoprotein (LDL) in the blood (D. T. Witiak and D. R Feller (Eds.), Anti-Lipidemic Drugs: Medicinal, Chemical and Biochemical Aspects, Elsevier, pp 159-195 (1991)). The formation of foam cells in the arterial wall increases with the ACAT activity. Accordingly, as gallic acid inhibits the action of ACAT, it may also lead to prevention of atherosclerosis and hyperlipidemia.
P-selectin is a key mediator in a variety of inflammatory processes and is implicated in, for instance, atherosclerosis and thrombosis. Therefore, the blocking of P-selectin is an attractive strategy for the treatment of these important diseases. Previously, a number of oligopeptides (with a consensus sequence Trp-Val-Asp-Val) was identified as selective P-selectin antagonists, displaying low micromolar affinity (WO 03/020753). Moreover, nanomolar range inhibitors could be obtained by functionalising the N-terminus of this and similar sequences with a galloyl (3,4,5-trihydroxybenzoyl) group (non pre-published international patent application PCT/EP03/07260).
Notwithstanding the increased understanding of some of the major diseases including coronary artery disease and atherosclerosis, and the availability of new compounds and methods to control the above-mentioned diseases and conditions to some degree, there still is a need for further improvements, both with respect to potent compounds and to methods to control or antagonise the effects of P-selectin activation in humans and to methods which reduce the risk of developing diseases associated with P-selectin activity which are cost-effective, acceptable to large fractions of the population, safe and tolerable. Finally there is a need for improved methods which can be used for early diagnosis of conditions leading to P-selectin associated diseases.